Silicone G thin layer analysis of soluble sugar.

related topics . Thin Layer Analysis (TLC) Technology
First, Experimental Purpose 1. Understanding the principle of
thin layer analysis
and determining soluble sugars..2. Master the operation method of silicone G thin layer analysis.II, the experimental principlethin layer analysis (referred to as
TLC
) is a micro-rapid layering method, is in adsorbents or support agents evenly coated on the thin layer, so called thin layer analysis. In order to separate the parts of the sample to be analyzed, a suitable adsorbent must be selected. Silicone, alumina and polyamide are widely used adsorbents, and diatomite and cellulose are commonly used support agents in distributive analysis, which can be applied after adding a suitable adhesive to the adsorbent or support agent, allowing the thin layer to be attached to the glass plate.Silicone G is a silicone powder with an adhesive added, containing about 12% to 13% of the gypsum (CaS04), which absorbs some substances from the solution to its surface. It is used to absorb different substances, and then use the appropriate solvent system layer, so that different substances can be separated. For example, the speed at which sugar moves on the thin layer of silicone G is related to the
relative molecular mass
and hydroxyl of sugar, and after the appropriate solvent is expanded, the speed at which sugar moves on the sheet is ethyl sugar >than sugar> double sugar> three sugars. The sugar content of the various parts can be measured by quantitative determination of the sugar by removing the sugar from the silicone G by scraping off the thin layer with the silicone G on another end-colored spot that is similar to its position. Thin layer analysis has obvious advantages over other methods: short layering time, large sample dosage range (1 sm;g to 0.5g), convenient observation results, can separate a variety of
compounds
and so on, its sensitivity is 10 to 100 times higher than paper lithography, color method can even use corrosive color agent. And thin layer analysis method is convenient to operate, simple equipment, so thin layer analysis method is currently widely used., instruments and
reagents instruments
ovens
, hair dryers, thin-layer glass plates (20cm×20cm),
edotoxicity meters
.reagents 1. 1% sugar standard solution: take xycosaccharide, glucose, fructose and sucrose each l00mg, with 75% ethanol, respectively, 10mL, so that the concentration is 10mg/mL..2. 0.1mol/L boric acid (H3BO3) solution..3. Layering solvent: chloroform: methanol: 60:40 (volume ratio)..4. Aniline-dephenylamine-phosphoric acid colorants:take 1g of dhiphenylamine, 1mL of aniline and 5mL85% of phosphoric acid dissolved in 50mL of acetone..5. Ketone reagents:called 0.2g ketones and 1g thiophenidate in the
Beagg
, slowly add 100mL thick sulphuric acid, side by side stirring, dissolved yellow transparent solution. Store it in a brown bottle, preferably ready-to-use, in the refrigerator can be stored for 2 weeks four, operation step(i) silicone G thin sheet preparation take silicone G powder 30g, add 75mL 0.1mol/L boric acid solution, adjust and lay on a clean, flat glass plate, the sheet after laying in 100 degrees C drying. Once removed, it can be used and stored
in
drying container. The thin surface requires a flat, thin and uniform surface.(ii) The separation of sugar on the thin layer of silicone G select a prepared sheet piece, select 4 points in a straight line 1.5cm from the bottom edge, with a distance of 2cm. With capillary tubes on different sugar samples at 4 points, sample volume control in 5 to 10 s mu;g, speckled diameter of no more than 2mm, to be thin layer on the sample natural drying, the sheet is placed in a layer folding cylinder containing laminate solvent, from the bottom up layer, when the layer solvent reached the top of the thin plate about 1 cm when taking out the sheet, the front line to make a mark, drying at 60 degrees C 2 to 3h (or drying in the air), in addition to the solvent evenly sprayed with a layer of aniline-dephenylamine-phosphoric acid colorant, drying at 85 degrees C 10min, then a variety of sugars show different colors.(iii) Quantitative determination of sugartake 5 dry silicone G-plates separately, and place the same 5 samples at even intervals on each plate, i.e., dot each sheet with 1 and m;L, 2-mu;L, 3-m; L, 5 and L sugar standard liquid 5 points, the operation method is the same, to the front of the solution to reach the top of the sheet lcm when removed, volatile solvent, the middle of each plate 3 samples of the analysis site covered with glass, the left and right side layer folding part with color agent positioning. Depending on the position of the colored speck, scrape the silicone G from the middle 3 corresponding areas with the blade and place it
the
tube. Add 1mL distilled water to each of the above test tubes, immerse it at room temperature for 1 hour, take 1mL filter liquid plus 2mL ketone reagents,
heat
10min in a boiling water bath, cool down with blank sample zeroing, at a wavelength of 620nm to measure the anti-light value of each sugar A620.(iv) Results Analysis 1. Write down the position and color of the spots, calculate the RF value, and draw the map of the layer.
.